内皮屏障抗原在实验性大鼠脑出血后表达及对血脑屏障通透性的影响
|
摘要
目的
脑出血(Intracerebral hemorrhage, ICH)是指非外伤性脑实质出血,是一种神经系统常见病、多发病,其致残率和死亡率均很高,严重影响人类的健康和患者的生存质量。ICH后脑水肿是脑损伤加重的主要原因之一,如果诱发脑疝还可导致患者死亡。目前的治疗方法仅局限于脱水治疗如高渗性脱水剂和利尿剂、外科手术减压治疗等,尚无特效治疗。内皮屏障抗原(Endothelial Barrier Antigen, EBA)是一种主要在具有血脑屏障(Blood-brain barrier,BBB)特性的毛细血管内皮细胞上特异性表达,由相对分子量分别为32kDa、25kDa和23.5kDa三个亚基组成的三聚体蛋白质。EBA在各种原因(如脑缺血、蛛网膜下腔出血、脑外伤等)引起的脑水肿形成中均起着重要的作用。本研究利用立体定向技术向大鼠尾状核内注入50 u L自体股动脉血建立脑出血模型,用免疫组化法检测脑出血后的血肿周围脑组织EBA和Fibrinogen动态表达变化、BBB的通透性及脑水含量的变化,探讨EBA在ICH后是否在脑出血后血肿周围脑组织表达及分布；EBA在ICH后脑水肿形成中的作用机制；从而探讨在脑出血后继发损伤(主要指脑水肿)中的作用。
材料与方法
一、材料
1、动物分组
选择健康雄性Sprague Dawley大鼠150只,体重250-300g,随机分成(1)正常对照组(2)假手术对照组(3)生理盐水对照(4)脑出血实验组,(3)(4)组分别按6h、1d、3d、7d分为4个亚组,每个亚组15只大鼠。
2、主要仪器
动物头颅立体定位仪、电子分析天平、显微图像分析仪、紫外分光光度仪、微量进样器、高温干燥箱、组织匀浆机、电泳仪、凝胶成像分析系统。
3、主要试剂
伊文思兰、甲酰胺、EBA兔抗鼠多克隆抗体、Fibrinogen羊抗鼠多克隆抗体、即用型SABC免疫组化试剂盒、DAB试剂盒三羟甲基氨基甲烷(Tris)、丙烯酰胺(Acr)、甲醇。
4、大鼠脑出血模型制作
用微量注射器抽取70uL大鼠自体股动脉血。立即将大鼠俯卧位于脑立体定位仪上,于前囟前0.2mm,中线右旁3.0mm,进针约6mm(大鼠尾状核处),将大鼠自体股动脉血50uL缓慢推注入脑。生理盐水对照组注入50uL生理盐水。假手术对照组钻孔不进行注射。正常对照组不予任何处置。
5、标本制作
(1)取各组5只大鼠于术后相应时间点麻醉,断头取脑,进行脑组织水含量测定和BBB通透性测定。
(2)取各组5只大鼠于术后相应时间点麻醉开胸,迅速暴露心脏,4%多聚甲醛灌注固定,取脑,样本置于4%多聚甲醛外固定4小时,酒精脱水,二甲苯透明,浸蜡,包埋。连续冠状切片,片厚5um,进行切片HE染色和免疫组化染色。
(3)取各组5只大鼠于术后相应时间点麻醉,断头取脑,进行Fibrinogen蛋白Western Blot检测。
二、检测指标
1、脑组织水含量测定
取大鼠出血侧针孔前侧脑组织,按照干湿重法测定脑水含量,计算公式为：脑组织含水量(%)=(湿重-干重)／湿重×100%。以脑组织水含量代表脑水肿的程度。
2、BBB通透性测定
取大鼠出血侧针孔后侧水肿区脑组织,按Belayev法使用伊文思兰(Evens blue,EB)测定BBB通透性。用EB含量(OD/mg)表示BBB通透性。
3、EBA蛋白、Fibrinogen蛋白表达免疫组化检测
SABC法进行EBA蛋白、Fibrinogen蛋白表达阳性细胞测定。切片经过热修复后滴加50uL一抗工作液(兔抗鼠多克隆EBA抗体,稀释度1：200、羊抗鼠多克隆Fibrinogen,稀释度1：50),4℃过夜；然后滴加生物素化山羊抗兔IgG,过氧化物酶标记兔抗羊IgG,37℃30min；滴加SABC,37℃30min,各步骤用PBS洗3 min×3次；DAB显色剂,苏木素轻度复染,脱水、透明、封片。显微图像分析系统采集图像,分析阳性细胞积分光密度。
4、Fibrinogen蛋白Western Blot检测
取出标本100mg低温提取蛋白,SDS-PAGE分离样品。转膜,封闭,加一抗(羊抗鼠多克隆Fibrinogen,稀释度1：100)孵育,洗涤,加二抗后再孵育,洗涤,显色,摄片。
三、统计分析
所有数据以均数±标准差(X±SD)表示,采用SPSS 16.0及Excel统计软件进行数据处理,组间比较用单因素方差分析(ANOVA),在单因素方差分析有意义的基础上再进行组间两两比较,两变量之间相关关系行Spearman相关分析,P<0.05为差异显著。
结果
1、脑水含量测定
大鼠实验性ICH后脑水含量均增高,6h开始升高,3d时达高峰,7d时逐渐降低,但是与对照组相比仍有显著差异(P<0.05)。
2、脑组织EB含量测定
大鼠实验性ICH后脑组织EB的含量增加,6h时即明显升高,3d时达到最高,7d组逐渐降低,但是与对照组相比仍有显著差异(P<0.05)。
3、大鼠实验性ICH后EBA蛋白表达
正常对照组大鼠皮质微血管内皮细胞膜EBA呈强阳性表达,假手术对照组和生理盐水对照组。脑出血后6h组在血肿周围和同侧皮质微血管内皮细胞膜EBA表达减少：脑出血后1d组EBA表达水平明显减少；脑出血3d组EBA表达显著减少；脑出血后7d组较3d组的EBA表达增加,但是仍低于6h和1d组。
4、大鼠实验性ICH后微血管周围Fibrinogen蛋白表达
正常对照组大鼠皮质微血管周围未见Fibrinogen表达,假手术对照组和生理盐水对照组Fibrinogen表达升高,但与正常组比较无显著差异。脑出血后6h在血肿周围大脑皮质有Fibrinogen弱阳性表达,阳性部位主要为微血管周围。1d组Fibrinogen表达水平明显增加。脑出血后3d达高峰,着色最深。7d后Fibrinogen表达水平逐渐降低,但较对照组仍有显著差异(P<0.05)。
5、Western blot检测Fibrinogen蛋白表达
正常对照组几乎未见Fibrinogen表达,假手术对照组和生理盐水对照组Fibrinogen表达与正常组比较无显著差异。脑出血后各组均可在34KD处见Fibrinogen蛋白表达条带。6h组有Fibrinogen弱阳性表达,1d和3d组Fibrinogen表达逐渐增加,3d达高峰,7d时Fibrinogen表达水平逐渐降低,但较对照组仍有显著差异(P<0.05)。
讨论
在本实验中采用向大鼠右侧尾状核内注射自体股动脉血制作脑出血模型,通过免疫组化法对EBA进行检测,发现出血后的大鼠脑组织中EBA的动态表达。另外,本实验对ICH后BBB的通透性分别采用外源性示踪剂(EB)和内源性示踪剂(Fibrinogen)两种方法进行了测量,进一步证实内源性示踪剂的准确性和可靠性。近年来,许多实验研究都证实,EBA参与了各种原因引发的脑水肿的发生和发展。本试验结果示,脑出血组与对照组相比,EBA的表达在6h开始减少,1d和3d组EBA表达水平逐渐减少,3d时EBA表达最少,以后表达逐渐恢复,7d组较前有恢复。而BBB通透性的变化规律与脑水含量的测定则与之相反,ICH后6h可见轻度升高,1d-3d逐渐升高,3d时达到高峰,之后逐渐下降,7d时仍高于正常水平。即BBB通透性和脑水肿的程度呈正相关,EBA的表达与BBB通透性和脑水肿的程度呈负相关。提示ICH后EBA在脑组织表达的减少、BBB通透性的开放均参与了脑水肿的形成。同时,BBB通透性与EBA的表达之间存在负性相关,提示EBA的表达减少可能通过影响BBB的通透性间接参与了脑水肿的形成。本文关于EBA在ICH后脑水肿病理生理机制的探讨结果,一方面完善ICH后脑水肿的发生和发展的可能机制,另一方面为临床探索有效治疗ICH的方法提供了一定的理论基础。
结论
1、本实验结果提示在大鼠脑出血后EBA在脑组织表达减少。
2、EBA可能通过影响BBB的通透性,参与脑出血后脑水肿的发生。
Objective
Intracerebral hemorrhage (ICH) is defined as non-traumatic cerebral parenchymal hemorrhage, which is a kind of disease commonly and frequently occurred in the nervous system. ICH has both high disability and fatality and greatly threatens the health of the patient and the quality of life. Post hemorrhage cerebral edema plays an important role in the deterioration of clinical condition. The cerebral brain hernia induced by it can also lead to death of patients. Treatment options were limited to osmotic agents such as hypertonic dehydration agents and diuretics, surgical decompression, non of which correct the molecular-level mechanisms responsible for brain edema. Endothelial Barrier Antigen (EBA) is a protein triplet of molecular weights 30,25, and 23.5 kDa located in the endothelial plasma membrane of cerebral microvessels, which is selectively expressed by endothelial cells (ECs) of rat blood-brain barrier (BBB). EBA is the key to the cerebral edema that is caused by cerebral ischemia, subarachnoid hemorrhage, brain trauma and etc. In this research, Horseley-Clarke technique is used to inject 50μL of autogenetic blood from femoral artery into caudate nucleus of mouses, immunohistochemistry is used to measure the expression levels of EBA and Fibrinogen, western blot is used to observe the expression of Fibrinogen, permeability of blood-brain barrier(BBB) and brain water content in rats, which aims to find out whether EBA is expressed and distributed in the brain tissue around hematoma, and then to discuss the role of EBA pathway in secondary lesion after cerebral hemorrhage.
Materials and methods
Materials
1. grouping
150 male Sprague Dawley mouses that weigh from 250 to 300g were randomly grouped into normal control group, sham-operated control group, saline control group, experimental group. The last two groups were respectively sub-grouped by 6h,1d,3d and 7d, n=15.
2. major equipments
Experimental cranial sterotaxic apparatus, electronic analytical balance, micro-image analysis apparatus, ultraviolet spectrometer, microsyringe, pyro-drying cabinet, tissue homogenater, electronic trophoresis apparatus, and gel-image analysis system.
3. major reagent
Evens blue, formamide, EBA rabbit anti-mouse polyclonal antibody, Fibrinogen sheep anti-mouse polyclonal antibody, instant SABC immunohistochemistry test kit, DAB test kit, Tris, Acr, and methanol.
4. models of cerebral hemorrhage
Models of slow injection with autoblood. Mouses were drawn 70uL arterial blood, put in prone position on stereotaxic apparatus, needled 0.2mm ahead of anterior fontanel,3.0mm right in median in depth of 6mm (caudate nucleus) and injected with 50uL arterial blood. Mouses in saline control group were injected with NS, Sham-operated control group without injection, while normal control group received no disposition.
5. making samples
(1)5 mouses were taken from each group after the corresponding time of operation, and anesthetized, decapitated and got brain off for brain water content and permeability of BBB.
(2) 5 mouses were taken from each group after the corresponding time of operation, immediately received heart exposition, infused with 4%paraform and got brains off. The taken sample were fixed in 4% paraform for 4 hours, dehydrated in alchohol, transparent by xylol, dipped in wax and embedded. Successive coronal-slice, about 5 um thick, and then stain.
(3)5 mouses were taken from each group after the corresponding time of operation, and anesthetized, decapitated and got brain off for Fibrinogen Western Blot detection。
Measure
1. brain water content
Anterior brain tissue of needle puncture was resectioned and determined by [(wet-dry weight)/wet weight]%. With brain water content as degree of brain edema.
2. permeability of BBB
Posterior lateral brain tissue of needle puncture was recetioned and assessed by Evens blue stain, as Belayev's method. Permeability of the BBB is assessed by EB content (OD/mg).
3. measuring EB A, Fibrinogen protein by immunohistochemistry
Use SABC to measure positive cells which express EBA, Fibrinogen protein. Add the slice with 50uL-antifluid (rabbit anti mouse polyclonal EBA antibody (1:200), sheep anti mouse polyclonal Fibrinogen antibody (1:50)), stay overnight at 4℃; add goat anti rabbit IgG, rabbit anti goat IgG at 37℃for 30min; add SABC at 37℃for 30min; every step washed by PBS for 3min 3 times; DAB developer, slightly restain by hematoxylin, dehydrate, transparent and mount. Use the microgram analysis system to collect images, and analyze spectrodensity of positive cells.
4. measuring Fibrinogen protein by.Western Blot
Remove the specimen 100mg, low-temperature extract proteins, SDS-PAGE separation of samples. Transfer film, closed, plus a goat anti-mouse polyclonal Fibrinogen(1:100), incubation bath, washing, add secondary antibody, then incubation bath, washing, color, photo film.
Statistical analysis
All datas were demonstrated by mean±standard deviation(x±SD), using SPSS 16.0 and Excel software for data processing and analysis of variance(ANOVA), comparing each two groups when statistically significant and using Spearman correlation analysis, P<0.05 when difference significant.
Result
1. brain water content
Experimental ICH in rats were significantly higher brain water content,6h began to increase,3d reached the peak,7d gradually reduced, as compared with the control group, but there are significant differences (P<0.05).
2. measure the EB content in brain tissue
After experimental ICH in rats brain the EB content increased,6h is significantly higher,3d reached the highest,7d group was gradually reduced, as compared with the control group, but there are significant differences(P<0.05).
3. expression of EBA after cerebral hemorrhage
Normal control rats cortex capillary endothelial cells showed strong positive expression of EBA, sham-operated control group and the saline control group compared with normal group there was no significant difference. After intracerebral hemorrhage, in 6h group the EBA expression in the ipsilateral cortex around the hematoma and endothelial cell membrane of micro vascular is decreased.1d EBA expression was significantly reduced; 3d the EBA expression was significantly reduced; the degree of EBA expression in 7d was increased than 3d, but still lower than the 6h and 1d.
4. expression of Fibrinogen after cerebral hemorrhage
Normal control rats have no Fibrinogen expression in the surrounding cortex microvessels. The expression of Fibrinogen in Sham-operated control group and the saline control group increased, but compared with the normal group, there was no significant difference. In 6h group, the cerebral cortex around the hematoma had weakly positive expression of Fibrinogen, and the positive sites were mainly around the capillaries.1d Fibrinogen expression levels increased significantly;3d Fibrinogen expression level reached the peak, coloring the most; 7d Fibrinogen expression level gradually decreased, but compared with the control group there are significant differences(P<0.05).
5. western blot detection of protein expression Fibrinogen
Fibrinogen almost has no expression in normal control group, Sham-operated control group and the saline control group Fibrinogen expression compared with normal group there was no significant difference. After intracerebral hemorrhage Fibrinogen protein 34KD band can be found in each group.6h Fibrinogen weakly positive expression;ld and 3d group Fibrinogen expression gradually increased,3d reached the peak;7d the expression of Fibrinogen has gradually reduced, but compared with the control group there are significant differences(P<0.05).
Discussion
We injected autoblood from femoral artery into right caudate nucleus to make models of cerebral hemorrhage, used immunohistochemistry to measure EBA and found out that there was change of expression of EBA protein after cerebral hemorrhage. In addition, the experiment after the BBB permeability of ICH was measured in two ways, exogenous tracers (EB) and endogenous tracer (Fibrinogen). And further confirmed the accuracy and reliability of endogenous tracer. In recent years, many experimental studies have confirmed that, EBA participated in the occurrence and development of brain edema caused by a variety of reasons. The test results showed cerebral hemorrhage group and the control group, EBA expression began to decrease after 6h, 1d and 3d group gradually reduced EBA expression, when 3d the EBA expression of the least gradually restored expression later,7d group recovered than before. The changes of BBB permeability and brain water content are in the opposite. The BBB permeability and brain water content result can be seen:6h after ICH slightly elevated, ld-3d gradually increased,3d reached its peak, and then gradually decreased, and 7d was still higher than normal levels. BBB permeability and brain edema was positively correlated. The expression of EBA was negatively correlated with BBB permeability and the extent of brain edema. After ICH, the decrease of EBA expression in brain tissue and the openness of BBB permeability all participate in the pahtophysilosical mechanisms in cerebral edema post hemorrhage. At the same time, the BBB permeability and the expression of EBA are negatively associated, suggesting that the decrease of EBA expression may indirectly influence the BBB permeability involved in the formation of brain edema. On the one hand, the study on the pathophysiological mechanism about the expression of EBA in post ICH cerebral edema may improve the possible mechanisms of brain edema after ICH, on the other hand, it may provide a sufficient theoretical basis for the effective treatment of the ICH.
Conclusion
1. These results indicate that in the EBA expression in brain tissue of rates reduced after intracerebral hemorrhage.
2. EBA may influence the BBB permeability and participate in the pahtophysilosical mechanisms in cerebral edema post hemorrhage.
脑出血(Intracerebral hemorrhage, ICH)是指非外伤性脑实质出血,是一种神经系统常见病、多发病,其致残率和死亡率均很高,严重影响人类的健康和患者的生存质量。ICH后脑水肿是脑损伤加重的主要原因之一,如果诱发脑疝还可导致患者死亡。目前的治疗方法仅局限于脱水治疗如高渗性脱水剂和利尿剂、外科手术减压治疗等,尚无特效治疗。内皮屏障抗原(Endothelial Barrier Antigen, EBA)是一种主要在具有血脑屏障(Blood-brain barrier,BBB)特性的毛细血管内皮细胞上特异性表达,由相对分子量分别为32kDa、25kDa和23.5kDa三个亚基组成的三聚体蛋白质。EBA在各种原因(如脑缺血、蛛网膜下腔出血、脑外伤等)引起的脑水肿形成中均起着重要的作用。本研究利用立体定向技术向大鼠尾状核内注入50 u L自体股动脉血建立脑出血模型,用免疫组化法检测脑出血后的血肿周围脑组织EBA和Fibrinogen动态表达变化、BBB的通透性及脑水含量的变化,探讨EBA在ICH后是否在脑出血后血肿周围脑组织表达及分布；EBA在ICH后脑水肿形成中的作用机制；从而探讨在脑出血后继发损伤(主要指脑水肿)中的作用。
材料与方法
一、材料
1、动物分组
选择健康雄性Sprague Dawley大鼠150只,体重250-300g,随机分成(1)正常对照组(2)假手术对照组(3)生理盐水对照(4)脑出血实验组,(3)(4)组分别按6h、1d、3d、7d分为4个亚组,每个亚组15只大鼠。
2、主要仪器
动物头颅立体定位仪、电子分析天平、显微图像分析仪、紫外分光光度仪、微量进样器、高温干燥箱、组织匀浆机、电泳仪、凝胶成像分析系统。
3、主要试剂
伊文思兰、甲酰胺、EBA兔抗鼠多克隆抗体、Fibrinogen羊抗鼠多克隆抗体、即用型SABC免疫组化试剂盒、DAB试剂盒三羟甲基氨基甲烷(Tris)、丙烯酰胺(Acr)、甲醇。
4、大鼠脑出血模型制作
用微量注射器抽取70uL大鼠自体股动脉血。立即将大鼠俯卧位于脑立体定位仪上,于前囟前0.2mm,中线右旁3.0mm,进针约6mm(大鼠尾状核处),将大鼠自体股动脉血50uL缓慢推注入脑。生理盐水对照组注入50uL生理盐水。假手术对照组钻孔不进行注射。正常对照组不予任何处置。
5、标本制作
(1)取各组5只大鼠于术后相应时间点麻醉,断头取脑,进行脑组织水含量测定和BBB通透性测定。
(2)取各组5只大鼠于术后相应时间点麻醉开胸,迅速暴露心脏,4%多聚甲醛灌注固定,取脑,样本置于4%多聚甲醛外固定4小时,酒精脱水,二甲苯透明,浸蜡,包埋。连续冠状切片,片厚5um,进行切片HE染色和免疫组化染色。
(3)取各组5只大鼠于术后相应时间点麻醉,断头取脑,进行Fibrinogen蛋白Western Blot检测。
二、检测指标
1、脑组织水含量测定
取大鼠出血侧针孔前侧脑组织,按照干湿重法测定脑水含量,计算公式为：脑组织含水量(%)=(湿重-干重)／湿重×100%。以脑组织水含量代表脑水肿的程度。
2、BBB通透性测定
取大鼠出血侧针孔后侧水肿区脑组织,按Belayev法使用伊文思兰(Evens blue,EB)测定BBB通透性。用EB含量(OD/mg)表示BBB通透性。
3、EBA蛋白、Fibrinogen蛋白表达免疫组化检测
SABC法进行EBA蛋白、Fibrinogen蛋白表达阳性细胞测定。切片经过热修复后滴加50uL一抗工作液(兔抗鼠多克隆EBA抗体,稀释度1：200、羊抗鼠多克隆Fibrinogen,稀释度1：50),4℃过夜；然后滴加生物素化山羊抗兔IgG,过氧化物酶标记兔抗羊IgG,37℃30min；滴加SABC,37℃30min,各步骤用PBS洗3 min×3次；DAB显色剂,苏木素轻度复染,脱水、透明、封片。显微图像分析系统采集图像,分析阳性细胞积分光密度。
4、Fibrinogen蛋白Western Blot检测
取出标本100mg低温提取蛋白,SDS-PAGE分离样品。转膜,封闭,加一抗(羊抗鼠多克隆Fibrinogen,稀释度1：100)孵育,洗涤,加二抗后再孵育,洗涤,显色,摄片。
三、统计分析
所有数据以均数±标准差(X±SD)表示,采用SPSS 16.0及Excel统计软件进行数据处理,组间比较用单因素方差分析(ANOVA),在单因素方差分析有意义的基础上再进行组间两两比较,两变量之间相关关系行Spearman相关分析,P<0.05为差异显著。
结果
1、脑水含量测定
大鼠实验性ICH后脑水含量均增高,6h开始升高,3d时达高峰,7d时逐渐降低,但是与对照组相比仍有显著差异(P<0.05)。
2、脑组织EB含量测定
大鼠实验性ICH后脑组织EB的含量增加,6h时即明显升高,3d时达到最高,7d组逐渐降低,但是与对照组相比仍有显著差异(P<0.05)。
3、大鼠实验性ICH后EBA蛋白表达
正常对照组大鼠皮质微血管内皮细胞膜EBA呈强阳性表达,假手术对照组和生理盐水对照组。脑出血后6h组在血肿周围和同侧皮质微血管内皮细胞膜EBA表达减少：脑出血后1d组EBA表达水平明显减少；脑出血3d组EBA表达显著减少；脑出血后7d组较3d组的EBA表达增加,但是仍低于6h和1d组。
4、大鼠实验性ICH后微血管周围Fibrinogen蛋白表达
正常对照组大鼠皮质微血管周围未见Fibrinogen表达,假手术对照组和生理盐水对照组Fibrinogen表达升高,但与正常组比较无显著差异。脑出血后6h在血肿周围大脑皮质有Fibrinogen弱阳性表达,阳性部位主要为微血管周围。1d组Fibrinogen表达水平明显增加。脑出血后3d达高峰,着色最深。7d后Fibrinogen表达水平逐渐降低,但较对照组仍有显著差异(P<0.05)。
5、Western blot检测Fibrinogen蛋白表达
正常对照组几乎未见Fibrinogen表达,假手术对照组和生理盐水对照组Fibrinogen表达与正常组比较无显著差异。脑出血后各组均可在34KD处见Fibrinogen蛋白表达条带。6h组有Fibrinogen弱阳性表达,1d和3d组Fibrinogen表达逐渐增加,3d达高峰,7d时Fibrinogen表达水平逐渐降低,但较对照组仍有显著差异(P<0.05)。
讨论
在本实验中采用向大鼠右侧尾状核内注射自体股动脉血制作脑出血模型,通过免疫组化法对EBA进行检测,发现出血后的大鼠脑组织中EBA的动态表达。另外,本实验对ICH后BBB的通透性分别采用外源性示踪剂(EB)和内源性示踪剂(Fibrinogen)两种方法进行了测量,进一步证实内源性示踪剂的准确性和可靠性。近年来,许多实验研究都证实,EBA参与了各种原因引发的脑水肿的发生和发展。本试验结果示,脑出血组与对照组相比,EBA的表达在6h开始减少,1d和3d组EBA表达水平逐渐减少,3d时EBA表达最少,以后表达逐渐恢复,7d组较前有恢复。而BBB通透性的变化规律与脑水含量的测定则与之相反,ICH后6h可见轻度升高,1d-3d逐渐升高,3d时达到高峰,之后逐渐下降,7d时仍高于正常水平。即BBB通透性和脑水肿的程度呈正相关,EBA的表达与BBB通透性和脑水肿的程度呈负相关。提示ICH后EBA在脑组织表达的减少、BBB通透性的开放均参与了脑水肿的形成。同时,BBB通透性与EBA的表达之间存在负性相关,提示EBA的表达减少可能通过影响BBB的通透性间接参与了脑水肿的形成。本文关于EBA在ICH后脑水肿病理生理机制的探讨结果,一方面完善ICH后脑水肿的发生和发展的可能机制,另一方面为临床探索有效治疗ICH的方法提供了一定的理论基础。
结论
1、本实验结果提示在大鼠脑出血后EBA在脑组织表达减少。
2、EBA可能通过影响BBB的通透性,参与脑出血后脑水肿的发生。
Objective
Intracerebral hemorrhage (ICH) is defined as non-traumatic cerebral parenchymal hemorrhage, which is a kind of disease commonly and frequently occurred in the nervous system. ICH has both high disability and fatality and greatly threatens the health of the patient and the quality of life. Post hemorrhage cerebral edema plays an important role in the deterioration of clinical condition. The cerebral brain hernia induced by it can also lead to death of patients. Treatment options were limited to osmotic agents such as hypertonic dehydration agents and diuretics, surgical decompression, non of which correct the molecular-level mechanisms responsible for brain edema. Endothelial Barrier Antigen (EBA) is a protein triplet of molecular weights 30,25, and 23.5 kDa located in the endothelial plasma membrane of cerebral microvessels, which is selectively expressed by endothelial cells (ECs) of rat blood-brain barrier (BBB). EBA is the key to the cerebral edema that is caused by cerebral ischemia, subarachnoid hemorrhage, brain trauma and etc. In this research, Horseley-Clarke technique is used to inject 50μL of autogenetic blood from femoral artery into caudate nucleus of mouses, immunohistochemistry is used to measure the expression levels of EBA and Fibrinogen, western blot is used to observe the expression of Fibrinogen, permeability of blood-brain barrier(BBB) and brain water content in rats, which aims to find out whether EBA is expressed and distributed in the brain tissue around hematoma, and then to discuss the role of EBA pathway in secondary lesion after cerebral hemorrhage.
Materials and methods
Materials
1. grouping
150 male Sprague Dawley mouses that weigh from 250 to 300g were randomly grouped into normal control group, sham-operated control group, saline control group, experimental group. The last two groups were respectively sub-grouped by 6h,1d,3d and 7d, n=15.
2. major equipments
Experimental cranial sterotaxic apparatus, electronic analytical balance, micro-image analysis apparatus, ultraviolet spectrometer, microsyringe, pyro-drying cabinet, tissue homogenater, electronic trophoresis apparatus, and gel-image analysis system.
3. major reagent
Evens blue, formamide, EBA rabbit anti-mouse polyclonal antibody, Fibrinogen sheep anti-mouse polyclonal antibody, instant SABC immunohistochemistry test kit, DAB test kit, Tris, Acr, and methanol.
4. models of cerebral hemorrhage
Models of slow injection with autoblood. Mouses were drawn 70uL arterial blood, put in prone position on stereotaxic apparatus, needled 0.2mm ahead of anterior fontanel,3.0mm right in median in depth of 6mm (caudate nucleus) and injected with 50uL arterial blood. Mouses in saline control group were injected with NS, Sham-operated control group without injection, while normal control group received no disposition.
5. making samples
(1)5 mouses were taken from each group after the corresponding time of operation, and anesthetized, decapitated and got brain off for brain water content and permeability of BBB.
(2) 5 mouses were taken from each group after the corresponding time of operation, immediately received heart exposition, infused with 4%paraform and got brains off. The taken sample were fixed in 4% paraform for 4 hours, dehydrated in alchohol, transparent by xylol, dipped in wax and embedded. Successive coronal-slice, about 5 um thick, and then stain.
(3)5 mouses were taken from each group after the corresponding time of operation, and anesthetized, decapitated and got brain off for Fibrinogen Western Blot detection。
Measure
1. brain water content
Anterior brain tissue of needle puncture was resectioned and determined by [(wet-dry weight)/wet weight]%. With brain water content as degree of brain edema.
2. permeability of BBB
Posterior lateral brain tissue of needle puncture was recetioned and assessed by Evens blue stain, as Belayev's method. Permeability of the BBB is assessed by EB content (OD/mg).
3. measuring EB A, Fibrinogen protein by immunohistochemistry
Use SABC to measure positive cells which express EBA, Fibrinogen protein. Add the slice with 50uL-antifluid (rabbit anti mouse polyclonal EBA antibody (1:200), sheep anti mouse polyclonal Fibrinogen antibody (1:50)), stay overnight at 4℃; add goat anti rabbit IgG, rabbit anti goat IgG at 37℃for 30min; add SABC at 37℃for 30min; every step washed by PBS for 3min 3 times; DAB developer, slightly restain by hematoxylin, dehydrate, transparent and mount. Use the microgram analysis system to collect images, and analyze spectrodensity of positive cells.
4. measuring Fibrinogen protein by.Western Blot
Remove the specimen 100mg, low-temperature extract proteins, SDS-PAGE separation of samples. Transfer film, closed, plus a goat anti-mouse polyclonal Fibrinogen(1:100), incubation bath, washing, add secondary antibody, then incubation bath, washing, color, photo film.
Statistical analysis
All datas were demonstrated by mean±standard deviation(x±SD), using SPSS 16.0 and Excel software for data processing and analysis of variance(ANOVA), comparing each two groups when statistically significant and using Spearman correlation analysis, P<0.05 when difference significant.
Result
1. brain water content
Experimental ICH in rats were significantly higher brain water content,6h began to increase,3d reached the peak,7d gradually reduced, as compared with the control group, but there are significant differences (P<0.05).
2. measure the EB content in brain tissue
After experimental ICH in rats brain the EB content increased,6h is significantly higher,3d reached the highest,7d group was gradually reduced, as compared with the control group, but there are significant differences(P<0.05).
3. expression of EBA after cerebral hemorrhage
Normal control rats cortex capillary endothelial cells showed strong positive expression of EBA, sham-operated control group and the saline control group compared with normal group there was no significant difference. After intracerebral hemorrhage, in 6h group the EBA expression in the ipsilateral cortex around the hematoma and endothelial cell membrane of micro vascular is decreased.1d EBA expression was significantly reduced; 3d the EBA expression was significantly reduced; the degree of EBA expression in 7d was increased than 3d, but still lower than the 6h and 1d.
4. expression of Fibrinogen after cerebral hemorrhage
Normal control rats have no Fibrinogen expression in the surrounding cortex microvessels. The expression of Fibrinogen in Sham-operated control group and the saline control group increased, but compared with the normal group, there was no significant difference. In 6h group, the cerebral cortex around the hematoma had weakly positive expression of Fibrinogen, and the positive sites were mainly around the capillaries.1d Fibrinogen expression levels increased significantly;3d Fibrinogen expression level reached the peak, coloring the most; 7d Fibrinogen expression level gradually decreased, but compared with the control group there are significant differences(P<0.05).
5. western blot detection of protein expression Fibrinogen
Fibrinogen almost has no expression in normal control group, Sham-operated control group and the saline control group Fibrinogen expression compared with normal group there was no significant difference. After intracerebral hemorrhage Fibrinogen protein 34KD band can be found in each group.6h Fibrinogen weakly positive expression;ld and 3d group Fibrinogen expression gradually increased,3d reached the peak;7d the expression of Fibrinogen has gradually reduced, but compared with the control group there are significant differences(P<0.05).
Discussion
We injected autoblood from femoral artery into right caudate nucleus to make models of cerebral hemorrhage, used immunohistochemistry to measure EBA and found out that there was change of expression of EBA protein after cerebral hemorrhage. In addition, the experiment after the BBB permeability of ICH was measured in two ways, exogenous tracers (EB) and endogenous tracer (Fibrinogen). And further confirmed the accuracy and reliability of endogenous tracer. In recent years, many experimental studies have confirmed that, EBA participated in the occurrence and development of brain edema caused by a variety of reasons. The test results showed cerebral hemorrhage group and the control group, EBA expression began to decrease after 6h, 1d and 3d group gradually reduced EBA expression, when 3d the EBA expression of the least gradually restored expression later,7d group recovered than before. The changes of BBB permeability and brain water content are in the opposite. The BBB permeability and brain water content result can be seen:6h after ICH slightly elevated, ld-3d gradually increased,3d reached its peak, and then gradually decreased, and 7d was still higher than normal levels. BBB permeability and brain edema was positively correlated. The expression of EBA was negatively correlated with BBB permeability and the extent of brain edema. After ICH, the decrease of EBA expression in brain tissue and the openness of BBB permeability all participate in the pahtophysilosical mechanisms in cerebral edema post hemorrhage. At the same time, the BBB permeability and the expression of EBA are negatively associated, suggesting that the decrease of EBA expression may indirectly influence the BBB permeability involved in the formation of brain edema. On the one hand, the study on the pathophysiological mechanism about the expression of EBA in post ICH cerebral edema may improve the possible mechanisms of brain edema after ICH, on the other hand, it may provide a sufficient theoretical basis for the effective treatment of the ICH.
Conclusion
1. These results indicate that in the EBA expression in brain tissue of rates reduced after intracerebral hemorrhage.
2. EBA may influence the BBB permeability and participate in the pahtophysilosical mechanisms in cerebral edema post hemorrhage.
引文
1 Gong C, Hoff JT, Keep RF, et al. Acute inflammatory reaction following experimental intracerebral hemorrhage in rat. Brain Research,2000,871 (4):57-65.
2 Lafuente JV, Argandona EG, and Mitre B.VEGFR-2 expression in brain injury:its distribution related to brain-blood barrier markers.Neural Transm,2006,113:487-496.
3 Hofman P, Hoyng P, van der Werf F, et al. Lack of blood-brain barrier properties in microvessels of the prelaminar optic nerve head.Invest Ophthalmol Vis Sci,2001,42:893-901.
4 Argandona EG, Bengoetxea H, Lafuente JV. Lack of experience-mediated differences in the immunohistochemical expression of blood-brain barrier markers (EB A and GluT-1)during the postnatal development of the rat visual cortex..Brian Res,2005,156:158-166.
5 Ebba Norsted, Burcak Gomuc, Bjorn Meister. Protein components of the blood-brain barrier in the mediobasal hypothalamus. J of Chemical Neuroanatomy,2008,36:107-121.
6 Belayev L, Busto R, Zhao W, et al. Quantitative evaluation of blood-brain barrier permeability following middle cerebral artery occlusion in rats.Brain Research, 1996,739(1-2):88-96.
7 Gong Y,Hua Y, Keep RF, et al. Intracerebral hemorrhage effects of aging on brain edema and neurologicaldeficits.Stroke,2004,35(11):2571-2575.
8 XueM, Balasubramaniam J, Del BigioMR. Brain inflammation following intracerebral hemorrhage. CurrNeuro-phar,2003,1(4):325-332.
9刘春梅,吴家名,等.脑出血后继发血管源性脑水肿的机制研究.皖南医学院学报,2003,22：171-173.
10 Lee KR. Colon GP, Betz L, et al. Edema from intracerebral hemorrhage:the role of thrombin. J Neurosurg,1996,84 (1):91-96.
11 William M.Blood-Brain Barrier Genomics.Stroke,2007,38:686-690.
12 Aoki T, Sumii T, Mori T, et al. Blood-brain barrier disruption and matrix metalloproteinase-9 expression during reperfusion injury:mechanical versus embolic focal ischemia in spontaneously hypertensive rats. Stroke,2002,33(11):2771-2717.
13 Ghabriel MN, Tadros R, Hermanis G, et al. A narrow time-window for access to t he brain by exogenous protein after immunological targeting of a blood-brain barrier antigen. J Comp Pat hol,2004,131:52-60.
14刘式威,尹岭,吕霞,等.动态观察糖尿病大鼠内皮屏障抗原和血脑屏障通透性的影响.中国神经精神疾病杂志,2007,33(5)：298-300.
15尹岭,刘式威,吕霞,等.实验性糖尿病大鼠内皮屏障抗原表达和血脑屏障通透性变化.中华神经科杂志,2007,40(1)：47-49.
16 Xuesong Chen, Jeremy W Gawryluk, John F Wagener, et al. Caffeine blocks disruption of blood brain barrier in a rabbit model of Alzheimer's disease.Journal of Neuroinflammation,2008,5:12-25.
17 Ryu JK, Davalos D, Akassoglou K. Fibrinogen signal transduction in the nervous system.J Thromb Haemost 2009,7 (Suppl.1):151-154.
18季耀东,陈衔城,夏鹰,等.重组链激酶早期应用对脑出血血肿周围脑组织的影响.复旦学报,2003,30(1)：30-33.
19 Nishigaya K, Yagi S, Sato T, et al. Impairment and restoration of the endothelial blood-brain barrier in the rat cerebral infarction model assessed by expression of endothelial barrier antigen immunoreactivity.Acta Neuropathol,2000,99:231-237.
20 Lin B, Ginsberg MD, Zhao W, et al. Quantitative Analysis of Microvascular Alterations in Traumatic Brain Injury by Endothelial Barrier Antigen Immunohistochemistry. J Neurtrauma, 2001,18:389-397.
21柯常庶,潘伟生,吴浩强.大鼠实验性脑创伤挫伤灶内皮屏障抗原和Ⅳ型水通道蛋白免疫反应性的动态改变.中国临床神经外科杂志,2004,9(1)：37-40.
22 Omura K, Ohbayashi M, Sano M, et al. The recovery of blood-nerve barrier in crush nerve injury-a quantitative analysis utilizing immunohistochemistry.Brain Research,2004, 1001:13-21.
23 Ghabriel MN, Zhu C, Hermanis G, et al. Immunological targeting of the endothelial barrier antigen (EBA) in vivo leads to opening of the blood-brain barrier.Brain Research,2000,878:127-35.
24 Ghabriel MN, Zhu C, Leigh C. Electron microscope study of blood-brain barrier opening induced by immunological targeting of the endothelial barrier antigen. Brain Research,2002, 934:140-151.
1 Gebel JM,Brott TG. Sila CA, et al. Decreased perihematomal edema in thrombolysis— related intracerebral hemorrhage compared with spontaneous intracerebral hemorrhage. Stroke,2000,31:596—600.
2吴家幂,刘春梅,李君.脑出血后周边组织继发性脑损伤炎性机制的研究.临床神经病学杂志，2004,17：278.
3 Rosenberg GA, Navratil W. Metalloproteinase inhibition blocksedem in intracerebral hemorrhage in the rat.Neurology,1997,48:921-926.
4 Gursoy-Ozdemir Y, Qiu J, Matsuoka N, et al. Cortical spreading depression activates and upregulates MMP-9.Clin Invest,2004,113:1447-1455.
5 Kim GW,Gasche Y,Grzeschik S,et al.Neurodegeneration in striatum induced by the mitochondrial toxin3-nitropropionic acid:Roleof matrix metalloproteinase-9 in early blood-brain barrier disrup-tion.Neuro Science,2003,23:8733-8742.
6吴家幂,刘胜达.实验性大鼠脑出血后脑组织MMP-9的表达及其意义.放射免疫学杂志,2005,18(5)：391-393.
7 Nie Benggang, Yu Ming, Li Xiaogang, et al. Relationship between MMP-9 and intracerebral hemorrhage injury.Med J West China,2007,19(5):767-769.
8 Wu CH, Huang FY, Wang KY,et al.Expression of matrix metalloproteinase MMP-9 in the plasma and hematoma fluid of intracerebral hemorrhage patients.,Zhonghua Yi Xue Za Zhi.2008,88(3):174-176.
9李燕华,孙善全.水通道蛋白-4在脑出血大鼠脑组织的分布.临床神经病学杂志,2004,17(2)：108-110.
10 Papadopoulos MC, Manley GT, Krishna S, et al. Aquaporin-4 facilitates reabsorption of excess fluid in vasogenic brain edema.FASEB J,2004,18(11):1291-1293.
11 Taniguchi M,Yamashita T,Kumura E,et al.Induction of aquaporin-4 water channel mRNA after focal cerebral ischemia in rat.Brain Res Mol Brain Res,2000,78(1-2):131-137.
12刘佳,王建平.大鼠实验性脑出血后血肿周围水通道蛋白-4动态变化的研究.国际神经病学神经外科学杂志,2006,33(1)：5-8.
13 Hiroaki Y,Tani K,Kamegawa A,et al.Implications of the Aquaporin-4 Structure on Array Formation and Cell Adhesion.J Mol.Biol,2006,355(4):628-639.
14 Guo FQ, Xu YC, Chen LY,et al.The relationship between the aquaporin-4 and brain edema,pathologic change,ultrastructure in perihematoma tissue in patients with intracerebral hemorrhage.Zhongguo Wei Zhong Bing Ji Jiu Yi Xue,2008,20(11):674-677.
15 Mayne M, Ni W, Yan HJ, et al. Antisense oligodeoxynucleotide inhibition of tumor necrosis factor-alpha expression is neuroprotecive after intracerebral hemorrhage.Stroke,2001,
32(1):240-248.
16 Hua Y,Wu J,Keep RF,et al. Tumor necrosis factor-alpha increases in the brain after intracerebral hemorrhage and thrombin stimulation.Neurosurgery,2006,58(3):542-50.
17史保中,范波,孟晓峰,等.红细胞对创伤性脑出血大鼠脑水含量与TNF-α表达的影响.郑州大学学报,2007,42(3)：506-509.
18 Xi G, Reiser G, Keep RF. The role of thrombin and thrombin receptors in ischemic, hemorrhagic and traumatic brain injury:deleterious or protective?. J Neurochem,2003,84 (1): 3-9.
19 Xi G, Keep R, Hoff J. Mechanism s of brain injury after intracerebral hemorrhage.Lancet Neurol,2006,5 (1):53-63.
20郑国庆,黄汉津,王小同.脑出血后凝血酶的作用与抗凝血酶治疗.国外医学脑血管疾病分册,2005,13(3)：199-201.
21 Coughlin SR.Thrombin signalling and proteaseactivated receptors.Nature,2000,407(4801): 258-264.
22关景霞,孙圣刚,曹学兵,等.凝血酶对血脑屏障通透性影响的实验研究.中风与神经疾病杂志,2003,20(4)：299-301.
23 Xi G, Hua Y, Keep RF. Induction of colligin may attenuate brain edema following intracerebral hemorrhage.Acta Neurochir Suppl,2000,76:501-505.
24 Hua Y, Keep RF, Hoff JT, et al. Thrombin preconditioning attenuates brain edema induced by erythrocytes and iron.J Cereb Blood Flow Metab,2003,23 (12):1448-1454.
25杨伟东,马景鑑.脑出血后脑组织凝血酶的表达与颅内压和预后的关系.中风与神经疾病杂志,2008,25(2)：219-221.
26 Homeister JW,Lucchesi BR.Complement activation and inhibition in myocardial ischemia and reperfusion injury.Annu Rev Pharmacol Toxicol,1994,34:17-40.
27吴家幂,马领松,周向阳,等.补体在脑出血后脑组织损伤机制中的作用.中华神经医学杂志,2006,5(2)：152-154.
28张军,张国华,林杰.补体C3和C4在脑出血后血肿周围组织水肿形成过程中的作用.中国全科医学,2008,11(5A)：741-743.
29 Ying Shan, Richard W,Lambrecht,et al.Role of Bach-1 in Regulation of Heme Oxygenase-1 in Human Liver Cells.J Biol Chem,2004,279(50):51769-51774.
30 W u J, Hua Y, Keep RF, et al. Iron and ironhandling p roteins in the brain after intracerebral hemorrhage.Stroke,2003,34 (12):2964-2969.
31 Wagner KR, Hua Y, de Courten-Myers GM, et al. Tin-mesoporphyrin, a potent hemeoxygenase inhibitor, for treatment of intracerebral hemorrhage:in vivo and in vitro studies.Cell Mol Biol,2000,46 (3):597-608.
32 Koeppen AH, Dickson AC, Smith J. Heme oxygenase in experimental intracerebral hemorrhage:the benefit of tin-mesoporphyrin Jneuropatho Exp Neuro,2004,63(6):587-597.
33 Gong Y, Tian H, Xi G, et al. Systemic zinc protoporphyrin administration reduces intracerebral hemorrhage-induced brain injury.Acta Neurochir Suppl,2006; 96:232-236.
34 Jian Wang, Sylvain Dore.Heme oxygenase-1 exacerbates early brain injury after intracerebral haemorrhage.Brain,2007,130:1643-1652.
35 Matsuda T,Arakawo N,Takuma K,et al.SEA0400,a novel and selective inhibitor of the Na+-Ca2+ exchanger, attenuates reperfusion injury in the vitro and in vivo cerebral ischemia models.J Pharmacol Exp Ther,2001,298:249-256.
36 Bano D, Nieotera P.Ca2+ signals and neuronaldeath in brain ischemia.Stroke,2007,38:674-676.
37 Ding XS. Feng MJ.Pathophsiology and apoptosis in ilschemic stroke.Int J Cerebrovase Dis, 2006,14:5-10.
38 Nilsson P, Hillered L, Olsson Y, et al. Regional change in interstitial K and Ca lever following cortical compression contusion trauma in rats.J Cereb Blood Flow Metab,1993:13(19):83.
39黄勇华,张微微,郝小淑,等.钙离子与鼠脑出血及继发性损伤的关系及尼莫地平的保护作用.中华老年心脑血管病杂志,2000,2(3)：200-202.
2 Lafuente JV, Argandona EG, and Mitre B.VEGFR-2 expression in brain injury:its distribution related to brain-blood barrier markers.Neural Transm,2006,113:487-496.
3 Hofman P, Hoyng P, van der Werf F, et al. Lack of blood-brain barrier properties in microvessels of the prelaminar optic nerve head.Invest Ophthalmol Vis Sci,2001,42:893-901.
4 Argandona EG, Bengoetxea H, Lafuente JV. Lack of experience-mediated differences in the immunohistochemical expression of blood-brain barrier markers (EB A and GluT-1)during the postnatal development of the rat visual cortex..Brian Res,2005,156:158-166.
5 Ebba Norsted, Burcak Gomuc, Bjorn Meister. Protein components of the blood-brain barrier in the mediobasal hypothalamus. J of Chemical Neuroanatomy,2008,36:107-121.
6 Belayev L, Busto R, Zhao W, et al. Quantitative evaluation of blood-brain barrier permeability following middle cerebral artery occlusion in rats.Brain Research, 1996,739(1-2):88-96.
7 Gong Y,Hua Y, Keep RF, et al. Intracerebral hemorrhage effects of aging on brain edema and neurologicaldeficits.Stroke,2004,35(11):2571-2575.
8 XueM, Balasubramaniam J, Del BigioMR. Brain inflammation following intracerebral hemorrhage. CurrNeuro-phar,2003,1(4):325-332.
9刘春梅,吴家名,等.脑出血后继发血管源性脑水肿的机制研究.皖南医学院学报,2003,22：171-173.
10 Lee KR. Colon GP, Betz L, et al. Edema from intracerebral hemorrhage:the role of thrombin. J Neurosurg,1996,84 (1):91-96.
11 William M.Blood-Brain Barrier Genomics.Stroke,2007,38:686-690.
12 Aoki T, Sumii T, Mori T, et al. Blood-brain barrier disruption and matrix metalloproteinase-9 expression during reperfusion injury:mechanical versus embolic focal ischemia in spontaneously hypertensive rats. Stroke,2002,33(11):2771-2717.
13 Ghabriel MN, Tadros R, Hermanis G, et al. A narrow time-window for access to t he brain by exogenous protein after immunological targeting of a blood-brain barrier antigen. J Comp Pat hol,2004,131:52-60.
14刘式威,尹岭,吕霞,等.动态观察糖尿病大鼠内皮屏障抗原和血脑屏障通透性的影响.中国神经精神疾病杂志,2007,33(5)：298-300.
15尹岭,刘式威,吕霞,等.实验性糖尿病大鼠内皮屏障抗原表达和血脑屏障通透性变化.中华神经科杂志,2007,40(1)：47-49.
16 Xuesong Chen, Jeremy W Gawryluk, John F Wagener, et al. Caffeine blocks disruption of blood brain barrier in a rabbit model of Alzheimer's disease.Journal of Neuroinflammation,2008,5:12-25.
17 Ryu JK, Davalos D, Akassoglou K. Fibrinogen signal transduction in the nervous system.J Thromb Haemost 2009,7 (Suppl.1):151-154.
18季耀东,陈衔城,夏鹰,等.重组链激酶早期应用对脑出血血肿周围脑组织的影响.复旦学报,2003,30(1)：30-33.
19 Nishigaya K, Yagi S, Sato T, et al. Impairment and restoration of the endothelial blood-brain barrier in the rat cerebral infarction model assessed by expression of endothelial barrier antigen immunoreactivity.Acta Neuropathol,2000,99:231-237.
20 Lin B, Ginsberg MD, Zhao W, et al. Quantitative Analysis of Microvascular Alterations in Traumatic Brain Injury by Endothelial Barrier Antigen Immunohistochemistry. J Neurtrauma, 2001,18:389-397.
21柯常庶,潘伟生,吴浩强.大鼠实验性脑创伤挫伤灶内皮屏障抗原和Ⅳ型水通道蛋白免疫反应性的动态改变.中国临床神经外科杂志,2004,9(1)：37-40.
22 Omura K, Ohbayashi M, Sano M, et al. The recovery of blood-nerve barrier in crush nerve injury-a quantitative analysis utilizing immunohistochemistry.Brain Research,2004, 1001:13-21.
23 Ghabriel MN, Zhu C, Hermanis G, et al. Immunological targeting of the endothelial barrier antigen (EBA) in vivo leads to opening of the blood-brain barrier.Brain Research,2000,878:127-35.
24 Ghabriel MN, Zhu C, Leigh C. Electron microscope study of blood-brain barrier opening induced by immunological targeting of the endothelial barrier antigen. Brain Research,2002, 934:140-151.
1 Gebel JM,Brott TG. Sila CA, et al. Decreased perihematomal edema in thrombolysis— related intracerebral hemorrhage compared with spontaneous intracerebral hemorrhage. Stroke,2000,31:596—600.
2吴家幂,刘春梅,李君.脑出血后周边组织继发性脑损伤炎性机制的研究.临床神经病学杂志，2004,17：278.
3 Rosenberg GA, Navratil W. Metalloproteinase inhibition blocksedem in intracerebral hemorrhage in the rat.Neurology,1997,48:921-926.
4 Gursoy-Ozdemir Y, Qiu J, Matsuoka N, et al. Cortical spreading depression activates and upregulates MMP-9.Clin Invest,2004,113:1447-1455.
5 Kim GW,Gasche Y,Grzeschik S,et al.Neurodegeneration in striatum induced by the mitochondrial toxin3-nitropropionic acid:Roleof matrix metalloproteinase-9 in early blood-brain barrier disrup-tion.Neuro Science,2003,23:8733-8742.
6吴家幂,刘胜达.实验性大鼠脑出血后脑组织MMP-9的表达及其意义.放射免疫学杂志,2005,18(5)：391-393.
7 Nie Benggang, Yu Ming, Li Xiaogang, et al. Relationship between MMP-9 and intracerebral hemorrhage injury.Med J West China,2007,19(5):767-769.
8 Wu CH, Huang FY, Wang KY,et al.Expression of matrix metalloproteinase MMP-9 in the plasma and hematoma fluid of intracerebral hemorrhage patients.,Zhonghua Yi Xue Za Zhi.2008,88(3):174-176.
9李燕华,孙善全.水通道蛋白-4在脑出血大鼠脑组织的分布.临床神经病学杂志,2004,17(2)：108-110.
10 Papadopoulos MC, Manley GT, Krishna S, et al. Aquaporin-4 facilitates reabsorption of excess fluid in vasogenic brain edema.FASEB J,2004,18(11):1291-1293.
11 Taniguchi M,Yamashita T,Kumura E,et al.Induction of aquaporin-4 water channel mRNA after focal cerebral ischemia in rat.Brain Res Mol Brain Res,2000,78(1-2):131-137.
12刘佳,王建平.大鼠实验性脑出血后血肿周围水通道蛋白-4动态变化的研究.国际神经病学神经外科学杂志,2006,33(1)：5-8.
13 Hiroaki Y,Tani K,Kamegawa A,et al.Implications of the Aquaporin-4 Structure on Array Formation and Cell Adhesion.J Mol.Biol,2006,355(4):628-639.
14 Guo FQ, Xu YC, Chen LY,et al.The relationship between the aquaporin-4 and brain edema,pathologic change,ultrastructure in perihematoma tissue in patients with intracerebral hemorrhage.Zhongguo Wei Zhong Bing Ji Jiu Yi Xue,2008,20(11):674-677.
15 Mayne M, Ni W, Yan HJ, et al. Antisense oligodeoxynucleotide inhibition of tumor necrosis factor-alpha expression is neuroprotecive after intracerebral hemorrhage.Stroke,2001,
32(1):240-248.
16 Hua Y,Wu J,Keep RF,et al. Tumor necrosis factor-alpha increases in the brain after intracerebral hemorrhage and thrombin stimulation.Neurosurgery,2006,58(3):542-50.
17史保中,范波,孟晓峰,等.红细胞对创伤性脑出血大鼠脑水含量与TNF-α表达的影响.郑州大学学报,2007,42(3)：506-509.
18 Xi G, Reiser G, Keep RF. The role of thrombin and thrombin receptors in ischemic, hemorrhagic and traumatic brain injury:deleterious or protective?. J Neurochem,2003,84 (1): 3-9.
19 Xi G, Keep R, Hoff J. Mechanism s of brain injury after intracerebral hemorrhage.Lancet Neurol,2006,5 (1):53-63.
20郑国庆,黄汉津,王小同.脑出血后凝血酶的作用与抗凝血酶治疗.国外医学脑血管疾病分册,2005,13(3)：199-201.
21 Coughlin SR.Thrombin signalling and proteaseactivated receptors.Nature,2000,407(4801): 258-264.
22关景霞,孙圣刚,曹学兵,等.凝血酶对血脑屏障通透性影响的实验研究.中风与神经疾病杂志,2003,20(4)：299-301.
23 Xi G, Hua Y, Keep RF. Induction of colligin may attenuate brain edema following intracerebral hemorrhage.Acta Neurochir Suppl,2000,76:501-505.
24 Hua Y, Keep RF, Hoff JT, et al. Thrombin preconditioning attenuates brain edema induced by erythrocytes and iron.J Cereb Blood Flow Metab,2003,23 (12):1448-1454.
25杨伟东,马景鑑.脑出血后脑组织凝血酶的表达与颅内压和预后的关系.中风与神经疾病杂志,2008,25(2)：219-221.
26 Homeister JW,Lucchesi BR.Complement activation and inhibition in myocardial ischemia and reperfusion injury.Annu Rev Pharmacol Toxicol,1994,34:17-40.
27吴家幂,马领松,周向阳,等.补体在脑出血后脑组织损伤机制中的作用.中华神经医学杂志,2006,5(2)：152-154.
28张军,张国华,林杰.补体C3和C4在脑出血后血肿周围组织水肿形成过程中的作用.中国全科医学,2008,11(5A)：741-743.
29 Ying Shan, Richard W,Lambrecht,et al.Role of Bach-1 in Regulation of Heme Oxygenase-1 in Human Liver Cells.J Biol Chem,2004,279(50):51769-51774.
30 W u J, Hua Y, Keep RF, et al. Iron and ironhandling p roteins in the brain after intracerebral hemorrhage.Stroke,2003,34 (12):2964-2969.
31 Wagner KR, Hua Y, de Courten-Myers GM, et al. Tin-mesoporphyrin, a potent hemeoxygenase inhibitor, for treatment of intracerebral hemorrhage:in vivo and in vitro studies.Cell Mol Biol,2000,46 (3):597-608.
32 Koeppen AH, Dickson AC, Smith J. Heme oxygenase in experimental intracerebral hemorrhage:the benefit of tin-mesoporphyrin Jneuropatho Exp Neuro,2004,63(6):587-597.
33 Gong Y, Tian H, Xi G, et al. Systemic zinc protoporphyrin administration reduces intracerebral hemorrhage-induced brain injury.Acta Neurochir Suppl,2006; 96:232-236.
34 Jian Wang, Sylvain Dore.Heme oxygenase-1 exacerbates early brain injury after intracerebral haemorrhage.Brain,2007,130:1643-1652.
35 Matsuda T,Arakawo N,Takuma K,et al.SEA0400,a novel and selective inhibitor of the Na+-Ca2+ exchanger, attenuates reperfusion injury in the vitro and in vivo cerebral ischemia models.J Pharmacol Exp Ther,2001,298:249-256.
36 Bano D, Nieotera P.Ca2+ signals and neuronaldeath in brain ischemia.Stroke,2007,38:674-676.
37 Ding XS. Feng MJ.Pathophsiology and apoptosis in ilschemic stroke.Int J Cerebrovase Dis, 2006,14:5-10.
38 Nilsson P, Hillered L, Olsson Y, et al. Regional change in interstitial K and Ca lever following cortical compression contusion trauma in rats.J Cereb Blood Flow Metab,1993:13(19):83.
39黄勇华,张微微,郝小淑,等.钙离子与鼠脑出血及继发性损伤的关系及尼莫地平的保护作用.中华老年心脑血管病杂志,2000,2(3)：200-202.